Leung E, Jacobson K A, Green R D
Department of Pharmacology, College of Medicine, University of Illinois, Chicago 60680.
Mol Pharmacol. 1990 Jul;38(1):72-83.
Previous work from our laboratory using sucrose gradient centrifugation and the antagonist radioligand [3H]xanthine amine congener led us to propose that A1 adenosine receptors are coupled to a GTP-binding protein (G protein) in the absence of an agonist and that adenosine receptor antagonists bind to free uncoupled receptors with high affinity and coupled receptors with low affinity and cause a destabilization of receptor-G protein complexes [Mol. Pharmacol. 36:412-419 (1989)]. Because agonists form high affinity ternary complexes composed of the agonist, receptor, and G protein, this hypothesis would imply that interactions between adenosine receptor agonists and antagonists, while competitive, would appear to be "noncompetitive" in nature. Interactions between unlabeled and radiolabeled A1 receptor agonist and antagonist ligands have been investigated using bovine cerebral cortical membranes to further probe this point. The availability of both 3H- and 125I-radioligands allowed us to use both single- and dual-isotope experimental designs. Radioligand antagonist-agonist competition curves along with saturation analyses using filtration and centrifugation to isolate bound radioligand suggested that agonists bind to two sites or receptor states with high affinity and to one site with low affinity. Agonist radioligand saturation curves with or without unlabeled antagonist suggested that antagonists do not bind to all states of the receptor with equal affinity. The computer program EQUIL was used to define models capable of simultaneously fitting all parts of complex experiments in which 125I-N6-aminobenzyladenosine saturation isotherms with or without 8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) and a saturation isotherm of [3H]CPX were performed. The data were not compatible with two-independent site models or with ternary complex models involving one receptor and one G protein. The data were fit by a model involving one receptor and two G proteins and by a model involving two receptors and one G protein. Both models suggest that 1) a high percentage of the receptor(s) is coupled to a G protein in the absence of an agonist and 2) agonists stabilize whereas antagonists destabilize precoupled receptor-G protein complexes. Because of this, competitive interactions between A1 agonists and antagonists appear noncompetitive in nature.
我们实验室先前利用蔗糖梯度离心法和拮抗剂放射性配体[3H]黄嘌呤胺类似物开展的研究，使我们提出以下观点：在不存在激动剂的情况下，A1腺苷受体与一种鸟苷三磷酸结合蛋白（G蛋白）偶联，并且腺苷受体拮抗剂以高亲和力与游离的未偶联受体结合，而与偶联受体的结合亲和力较低，并导致受体 - G蛋白复合物的不稳定[《分子药理学》36:412 - 419(1989)]。由于激动剂形成由激动剂、受体和G蛋白组成的高亲和力三元复合物，该假设意味着腺苷受体激动剂与拮抗剂之间的相互作用虽然具有竞争性，但在本质上似乎是“非竞争性”的。已使用牛大脑皮层膜研究了未标记和放射性标记的A1受体激动剂与拮抗剂配体之间的相互作用，以进一步探究这一点。3H和125I放射性配体的可得性使我们能够采用单同位素和双同位素实验设计。放射性配体拮抗剂 - 激动剂竞争曲线以及使用过滤和离心分离结合的放射性配体的饱和分析表明，激动剂以高亲和力与两个位点或受体状态结合，以低亲和力与一个位点结合。有无未标记拮抗剂时的激动剂放射性配体饱和曲线表明，拮抗剂并非以同等亲和力与受体的所有状态结合。计算机程序EQUIL用于定义能够同时拟合复杂实验所有部分的模型，在这些实验中进行了有无8 - 环戊基 - 1,3 - 二丙基黄嘌呤（[3H]CPX）时的125I - N6 - 氨基苄基腺苷饱和等温线以及[3H]CPX的饱和等温线。数据与两个独立位点模型或涉及一个受体和一个G蛋白的三元复合物模型不相符。数据由涉及一个受体和两个G蛋白的模型以及涉及两个受体和一个G蛋白的模型拟合。两个模型均表明：1）在不存在激动剂的情况下，高比例的受体与G蛋白偶联；2）激动剂使预偶联的受体 - G蛋白复合物稳定，而拮抗剂使其不稳定化。因此，A1激动剂与拮抗剂之间的竞争性相互作用在本质上表现为非竞争性。
